miR-23a-27a-24簇在猪卵巢颗粒细胞凋亡中的作用  

The role of the miR-23a-27a-24 cluster in the apoptosis of porcine ovarian granulosa cells

作　　者：王思琪 张卓凡 杨柳 杜星[1] 吴望军[1] 李齐发[1] WANG Siqi;ZHANG Zhuofan;YANG Liu;DU Xing;WU Wangjun;LI Qifa(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物科技学院,江苏南京210095

出　　处：《畜牧与兽医》2022年第4期1-9,共9页Animal Husbandry & Veterinary Medicine

基　　金：江苏省农业重大新品种创新专项(PZCZ201734)。

摘　　要：为了解猪miR-23a-27a-24簇的序列及结构特征,分析其在猪卵巢颗粒细胞凋亡中的作用,采用测序技术获得猪miR-23a-27a-24簇前体序列;利用生物信息学方法分析miR-23a-27a-24簇的序列信息、结构和生物学功能;使用双酶切法构建猪miR-23a-27a-24簇的过表达载体,转染至体外培养的猪卵巢颗粒细胞中,qRT-PCR检测过表达效果;通过流式细胞术检测颗粒细胞凋亡率。结果显示:猪miR-23a-27a-24簇前体序列长度为408 bp,核苷酸序列与其他哺乳动物的一致性较高;哺乳动物miR-23a-27a-24簇均位于基因组中ZSWIM和miR-181c基因之间,成员转录顺序相同、间距相近;功能富集分析发现,miR-23a-27a-24簇的靶基因主要富集于RNA聚合酶Ⅱ启动子转录的正调控的生物学过程和PI3K-Akt信号通路中;成功构建猪miR-23a-27a-24簇的过表达载体,且在猪卵巢颗粒细胞中高效表达;流式细胞术分析发现,转染miR-23a-27a-24簇过表达载体能够显著提高颗粒细胞的凋亡率(P<0.05),极显著提高颗粒细胞早期凋亡率(P<0.01)。结果表明miR-23a-27a-24簇能够促进猪卵巢颗粒细胞凋亡。To understand the sequence and structural characteristics of the porcine miR-23 a-27 a-24 cluster, and analyze its role in the apoptosis of porcine ovarian granulosa cells, we used PCR amplification and DNA sequencing to obtain the precursor sequences of the miR-23 a-27 a-24 cluster in pigs, and used bioinformatics to analyze the sequence, structure and biological function of the cluster. The overexpression vector of the miR-23 a-27 a-24 cluster, which was constructed using the double enzyme digestion method, was transfected into porcine ovarian granulosa cells cultured in vitro. The mRNA expression levels of miR-23 a, miR-27 a and miR-24 were detected by quantitative real-time PCR. The apoptosis of the granulosa cells was detected by flow cytometry. The results showed that the precursor sequence of the porcine miR-23 a-27 a-24 cluster was 408 bp, which was highly consistent among mammals. Mammalian miR-23 a-27 a-24 clusters have the same transcription sequence and similar spacing between members, which are located between the ZSWIM and miR-181 c genes in the genome. Functional enrichment analysis showed that the target genes of the miR-23 a-27 a-24 cluster were mainly enriched in the biological process of positive regulation of RNA polymerase II promoter transcription and the PI3 K Akt signaling pathway. The overexpression vector of the porcine miR-23 a-27 a-24 cluster was successfully constructed, and it was highly expressed in the porcine ovarian granulosa cells. The total apoptotic rate and early apoptotic rate of granulosa cells were significantly increased(P<0.05) and extremely significantly increased(P<0.01) by transfection of the miR-23 a-27 a-24 cluster overexpression vector. The present results indicated that the miR-23 a-27 a-24 cluster promoted the apoptosis of porcine ovarian granulosa cells.

关 键 词：猪 卵巢颗粒细胞 细胞凋亡 miR-23a-27a-24簇 

分 类 号：S828[农业科学—畜牧学]